Compositions and methods for inhibiting expression of a mutant gene

ABSTRACT

The present invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a mutant gene, comprising a first strand that has a complementary region that is complementary to at least a portion of an RNA transcript of at least part of the mutant target gene and a second strand of the dsRNA complementary or substantially complementary to the first strand. The invention further relates to a pharmaceutical composition comprising the dsRNA and a pharmaceutically acceptable carrier. The pharmaceutical compositions are useful for inhibiting the expression of a target mutant gene, as well as for treating diseases caused by expression of the target gene. The invention also relates to methods for inhibiting the expression of a target mutant gene, as well as methods for treating diseases caused by the expression of the target gene.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/817,009, filed on Jun. 16, 2010, which is a divisional of U.S.application Ser. No. 10/384,463 (issued), filed on Mar. 7, 2003, whichis a continuation-in-part of International Application No.PCT/EP02/11969, which designated the United States and was filed on Oct.25, 2002, which claims the benefit of German Patent No. 101 55 280.7,filed on Oct. 26, 2001, German Patent No. 101 58 411.3, filed on Nov.29, 2001, German Patent No. 101 60 151.4, filed on Dec. 7, 2001,International Application No. PCT/EP02/00152, filed on Jan. 9, 2002,International Application No. PCT/EP02/00151, filed on Jan. 9, 2002, andGerman Patent No. 102 35 620.3, filed on Aug. 2, 2002. The entireteachings of the above applications are incorporated herein by referencein their entireties, including any appendices thereof, for all purposes.

FIELD OF THE INVENTION

This invention relates to compositions and methods for inhibiting theexpression of a mutant gene using double-stranded ribonucleic acid(dsRNA) to mediate RNA interference.

BACKGROUND OF THE INVENTION

Many genetic diseases and defects are caused by only a minor mutation ina specific gene, such as a single-base mismatch (see, e.g., Cooper, D.N., et al., in “The Metabolic and Molecular Bases of Inherited Disease”(Scriver, C. R., et al., eds., (McGraw-Hill Inc., New York, Vol. 1, pp.259-291 (1995)). Due to the high degree of sequence homology,therapeutic agents designed to inhibit the expression of a gene having asingle (or multiple) point mutation almost inevitably affects theexpression of the normal gene. Treating diseases that result from suchgenetic aberrations is problematic, particularly in proliferativediseases such as cancer, were expression of the non-mutated gene isessential for normal cellular function.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNAse III Dicer processes dsRNA intosmall interfering RNAs (siRNA) of approximately 22 nucleotides, whichserve as guide sequences to induce target-specific mRNA cleavage by anRNA-induced silencing complex RISC (Hammond, S. M., et al., Nature(2000) 404:293-296). When administered to a cell or organism, exogenousdsRNA has been shown to direct the sequence-specific degradation ofendogenous messenger RNA (mRNA) through RNAi. This phenomenon has beenobserved in a variety of organism, including mammals (see, e.g., WO00/44895, Limmer; and DE 101 00 586 C1, Kreutzer et al.).

While completely complementary dsRNA are robust inhibitors ofexpression, Elbashir et al. have shown that dsRNA having athree-nucleotide mismatch with the target gene are very poor mediatorsof RNA interference (Elbashir, S. M., et al., Nature (2001)411:494-498). On the other hand, Holen et al. have shown that dsRNAhaving one or two nucleotide mismatches can induce RNA interference,thereby inhibiting the expression of the target gene (Holen, T., et al.,Nucl. Acid Res. (2002) 1757-1766). Thus, there appears to be no cleardemarcation in activity upon which to design a dsRNA-based therapeuticagent for treating a disease resulting from a minor genetic aberration.Such an agent would likely produce serious side effects, due to thepotential cross-reactivity between the mutant gene and its normalcellular counterpart gene.

While dsRNA can effectively silence a specific target gene, there iscurrently no available means for selectively inhibiting the expressionof a gene comprising a point mutation without also inhibiting theexpression of the normal, non-mutated gene. Thus, there remains a needfor an agent that can selectively and efficiently silence a mutant genewithout also affecting its wild-type counterpart. Compositionscomprising such agents would be useful for treating genetic diseases anddisorders caused by the expression of a gene having a minor mutation,such as a single or multiple-base mismatch.

SUMMARY OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting theexpression of a target gene having a point mutation(s) using the dsRNA.The present invention also discloses compositions and methods fortreating diseases caused by the expression of a mutant gene. The dsRNAof the invention comprises an RNA strand (the complementary strand)having a complementary region that is substantially complementary to anRNA transcript of a target gene having a point mutation, and which ispartially complementary to the counterpart, non-mutated cellular gene.More specifically, the complementary region of the dsRNA has at leastone base-pair mismatch with the target mutant gene, and at least onemore base-pair mismatch with the non-mutated cellular gene.

In one aspect, the invention relates to a double-stranded ribonucleicacid (dsRNA) for selective inhibition of expression of a target mutantgene. The dsRNA comprises a complementary RNA strand and a sense RNAstrand, wherein the complementary RNA strand comprises a nucleotidesequence which is substantially complementary to at least a part of thetarget mutant gene, and partially complementary to at least a part of acorresponding wild-type gene. The complementary nucleotide region maycomprise a nucleotide mismatch with the target mutant gene, wherein themismatch is at least 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 6 nucleotides, 7, nucleotides, 8nucleotides, 9 nucleotides, or 10 nucleotides from either end of saidnucleotide sequence. The target mutant gene may comprise a pointmutation not present in the corresponding wild-type gene. Thecomplementary nucleotide region may have a one-nucleotide sequencemismatch with the target mutant gene, and a two-nucleotide mismatch withthe corresponding wild-type gene. The complementary RNA strand and thesense RNA strand may have a nucleotide mismatch. The complementary RNAstrand may comprise a 3′-end and a 5′-end, wherein one of the ends has anucleotide overhang of 1 to 4 nucleotides, preferably 2 or 3 nucleotidesin length. The nucleotide overhang is preferably at the 3′-end of thecomplementary RNA strand, and the 5′-end is blunt. The complementary RNAstrand may be 23 nucleotides in length and the sense RNA strand may be21 nucleotides in length. The complementary RNA strand may becomplementary to an RNA transcript of the target mutant gene. The targetmutant gene may be an oncogene.

In another aspect, the invention relates to a method for selectivelyinhibiting the expression of a target mutant gene in a cell. The methodcomprises introducing into the cell a dsRNA, as described above, andmaintaining the cell for a time sufficient to obtain the selectiveinhibition of expression of the target gene. The complementary RNAstrand comprises a nucleotide sequence which is substantiallycomplementary to at least a part of the target mutant gene, and which ispartially complementary to at least a part of a corresponding wild-typegene. The target mutant gene may be an oncogene.

In still another aspect, the invention relates to a pharmaceuticalcomposition for selectively inhibiting the expression of a target mutantgene in a mammal. The pharmaceutical composition comprises a dsRNA, asdescribed above, and pharmaceutically acceptable carrier. The dsRNAcomprises a complementary RNA strand and a sense RNA strand, wherein thecomplementary RNA strand comprises a nucleotide sequence which issubstantially complementary to at least a part of the target mutantgene, and which is partially complementary to at least a part of acorresponding wild-type gene. The dosage unit of dsRNA may be in a rangeof 0.01 to 5.0 milligrams, 0.01 to 2.5 milligrams, 0.1 to 200micrograms, 0.1 to 100 micrograms, 1.0 to 50 micrograms, or preferably1.0 to 25 micrograms per kilogram body weight of the mammal. The mammalmay be a human. The pharmaceutically acceptable carrier may be anaqueous solution, such as a phosphate buffered saline. Thepharmaceutically acceptable carrier may comprise a micellar structure,such as a liposome, capsid, capsoid, polymeric nanocapsule, or polymericmicrocapsule. The pharmaceutical composition may be formulated to beadministered by inhalation, infusion, injection, or orally, preferablyby intravenous or intraperitoneal injection.

In yet another aspect, the invention relates to a method for treating adisease caused by the expression of a target mutant gene in a mammal.The method comprises administering to the mammal a pharmaceuticalcomposition comprising a dsRNA and a pharmaceutically acceptablecarrier, both of which are described above. The dsRNA comprises acomplementary RNA strand and a sense RNA strand, wherein thecomplementary RNA strand comprises a nucleotide sequence which issubstantially complementary to at least a part of the target mutantgene, and also partially complementary to at least a part of acorresponding wild-type gene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of sequence complementarity on the inhibition ofexpression of an HCV luciferase fusion protein by dsRNAs.

FIG. 2 shows the effect of sequence complementarity, as well as theeffect of a single base-pair mismatch between RNA strands of the dsRNA,on the inhibition of expression of an HCV luciferase fusion protein bydsRNAs

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses double-stranded ribonucleic acid(dsRNA), as well as compositions and methods for inhibiting theexpression of a mutant gene in a cell using the dsRNA. The presentinvention also discloses pharmaceutical compositions and methods fortreating diseases in organisms caused by expression of a gene having atleast one point mutation using dsRNA. dsRNA directs thesequence-specific degradation of mRNA through a process known as RNAinterference (RNAi). The process occurs in a wide variety of organisms,including mammals and other vertebrates.

The dsRNA of the invention comprises an RNA strand (the complementarystrand) having a complementary region that is substantiallycomplementary to an RNA transcript of the target mutant gene, and whichis partially complementary to the counterpart, non-mutated cellulargene. The complementary region of the dsRNA has at least one base-pairmismatch with the target gene, and at least one more base-pair mismatchwith its non-mutated counterpart. Using a cell-based assay system, thepresent inventors have demonstrated the selective inhibition of a targetmutant gene (i.e., inhibition of the mutant gene without concomitantinhibition of expression of the corresponding wild-type gene) using adsRNA comprising at least a two-base pair mismatch with the wild-typegene. Moreover, the inventors have shown a direct relationship betweenthe extent of complementarity between the single strands of the dsRNAand the inhibitory activity of the dsRNA. Thus, one can modify theefficiency of inhibition by altering the degree of complementaritybetween the two strands of the dsRNA. The present invention encompassesthese dsRNAs and compositions comprising dsRNA and their use forspecifically silencing genes with point mutations. The use of thesedsRNAs enables the targeted degradation of mRNAs of mutated genes thatare implicated in a wide variety of disease processes, includingcellular proliferative disorders, while minimizing the affect on normalcellular activities. Thus, the methods and compositions of the presentinvention comprising these dsRNAs are useful for treating diseases anddisorders caused by the expression of a mutant gene.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of atarget mutant gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of the gene. Thepharmaceutical compositions of the present invention comprise a dsRNAhaving a region which is substantially complementary to an RNAtranscript of the target mutant gene and which is only partiallycomplementary to the counterpart non-mutated gene, together with apharmaceutically acceptable carrier. The complementary region of thedsRNA has at least a one base-pair mismatch with the target gene, and atleast one more base-pair mismatch with its non-mutated counterpart.Preferably, the complementary strand of the dsRNA is complementary to atleast a portion of the 3′-untranslated region of the mRNA. The base-pairmismatch(es) is preferably located at least at least 1 nucleotide, 2nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides,7, nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides awayfrom either end of the RNA strand.

Accordingly, certain aspects of the present invention relate topharmaceutical compositions comprising the dsRNA of the presentinvention together with a pharmaceutically acceptable carrier, methodsof using the compositions to inhibit expression of a target mutant gene,and methods of using the pharmaceutical compositions to treat diseasescaused by the expression of a gene having a point mutation.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below.

As used herein, the terms “target” refers to a section of a DNA strandof a double-stranded DNA that is complementary to a section of a DNAstrand, including all transcribed regions, that serves as a matrix fortranscription. A target gene, usually the sense strand, is a gene whoseexpression is to be selectively inhibited or silenced through RNAinterference.

As used herein, the terms “mutant gene” and “target mutant gene” referto a gene comprising at least one point mutation relative to thecorresponding normal, non-mutated cellular gene (referred to herein asthe “corresponding wild-type gene”). The terms mutant gene and targetmutant gene specifically encompass any variant of a normal cellular geneor gene fragment whose expression is associated with a disease ordisorder (e.g., an oncogene). Preferably, the target mutant gene has oneor two point mutations relative to the corresponding wild-type gene.

The term “complementary RNA strand” (also referred to herein as the“antisense strand”) refers to the strand of a dsRNA which iscomplementary to an mRNA transcript that is formed during expression ofthe target gene, or its processing products. As used herein, the term“complementary nucleotide sequence” refers to the region on thecomplementary RNA strand that is complementary to a region of an mRNAtranscript of the target mutant gene (i.e., “the correspondingnucleotide sequence” of the target gene). “dsRNA” refers to aribonucleic acid molecule having a duplex structure comprising twocomplementary and anti-parallel nucleic acid strands. Not allnucleotides of a dsRNA must exhibit Watson-Crick base pairs. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. The RNA strands may have the same or a different number ofnucleotides. The complementary nucleotide region of a complementary RNAstrand is less than 25, preferably 19 to 24, more preferably 20 to 24,even more preferably 21 to 23, and most preferably 22 or 23 nucleotidesin length. The complementary RNA strand is less than 30, preferablyfewer than 25, more preferably 21 to 24, and most preferably 23nucleotides in length. dsRNAs comprising a complementary or antisensestrand of this length (known as “short interfering RNA” or “siRNA”) areparticularly efficient in inhibiting the expression of the target mutantgene. “Introducing into” means uptake or absorption in the cell, as isunderstood by those skilled in the art. Absorption or uptake of dsRNAcan occur through cellular processes, or by auxiliary agents or devices.For example, for in vivo delivery, dsRNA can be injected into a tissuesite or administered systemically. In vitro delivery includes methodsknown in the art such as electroporation and lipofection.

As used herein, two polynucleotide sequences are said to be“substantially complementary” to each other when all but one of theirrespective nucleotides are capable of forming base pairs. The terms“nucleotide mismatch” and “mismatch” refer to a pair of bases that areincapable of forming base-pairs. As used herein, two polynucleotidesequences are said to be “partially complementary” to each other whenthey have at least one more nucleotide mismatch than their substantiallycomplementary counterparts (i.e., they share at least one fewer basepairs). For example, if polynucleotides A and B are substantiallycomplementary, then polynucleotides A and C are partially complementaryif all but at least two of their nucleotides are incapable of formingbase pairs. Preferably, the nucleotides that are incapable of formingbase pairs are located at least at least 1 nucleotide, 2 nucleotides, 3nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7,nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides away fromeither end of the nucleotide strand.

As used herein, “selective inhibition of expression” means that a dsRNAhas a greater inhibitory effect on the expression of a target mutantgene than on the corresponding wild-type gene. Preferably, theexpression level of the target mutant gene is less than 98%, less than95%, less than 90%, less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, or less than 10%of the expression level of the corresponding wild-type gene.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure when a3′-end of one RNA strand extends beyond the 5′-end of the other strand,or vice versa.

As used herein and as known in the art, the term “identity” is therelationship between two or more polynucleotide sequences, as determinedby comparing the sequences. Identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween strings of such sequences. Identity can be readily calculated(see, e.g., Computation Molecular Biology, Lesk, A. M., eds., OxfordUniversity Press, New York (1998), and Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York (1993),both of which are incorporated by reference herein). While there exist anumber of methods to measure identity between two polynucleotidesequences, the term is well known to skilled artisans (see, e.g.,Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J.,eds., M. Stockton Press, New York (1991)). Methods commonly employed todetermine identity between sequences include, for example, thosedisclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988)48:1073. “Substantially identical,” as used herein, means there is avery high degree of homology (preferably 100% sequence identity) betweenthe sense strand of the dsRNA and the corresponding part of the targetgene. However, dsRNA having greater than 90% or 95% sequence identitymay be used in the present invention, and thus sequence variations thatmight be expected due to genetic mutation, strain polymorphism, orevolutionary divergence can be tolerated. Although 100% identity ispreferred, the dsRNA may contain single or multiple base-pair randommismatches between the RNA and the target gene.

As used herein, the term “treatment” refers to the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disorder, e.g., a disease or condition, asymptom of disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)

In one embodiment, the invention relates to a double-strandedribonucleic acid (dsRNA) having a nucleotide sequence which issubstantially identical to at least a portion of a target mutant gene.The dsRNA comprises two RNA strands that are sufficiently complementaryto hybridize to form the duplex structure. One strand of the dsRNAcomprises the nucleotide sequence that is substantially identical to aportion of the target gene (the “sense” strand), and the other strand(the “complementary” or “antisense” strand) comprises a sequence that iscomplementary to an RNA transcript of the target mutant gene. The dsRNAcomprises an RNA strand (the complementary strand) having acomplementary region that is substantially complementary to an RNAtranscript of the target mutant gene, and which is partiallycomplementary to the counterpart wild-type gene. The complementaryregion of the dsRNA has at least one base-pair mismatch with the targetmutant gene, and at least one more base-pair mismatch with thecorresponding non-mutated (wild-type) gene. The dsRNA has less than 30nucleotides, preferably less than 25 nucleotides, more preferablybetween 21 and 24 nucleotides, and most preferably 23 nucleotides inlength. The dsRNA can be synthesized by standard methods known in theart, e.g., by use of an automated DNA synthesizer, such as arecommercially available from Biosearch, Applied Biosystems, Inc.

In one embodiment, the dsRNA is designed to provide a minimal risk ofcross-reactivity with normal cellular genes, and thus minimal sideeffects. In this embodiment, the dsRNA has the maximum number ofmismatches between the complementary nucleotide sequence of thecomplementary RNA strand and the corresponding nucleotide sequence ofthe target mutant gene that are permissible, while still providing adetectable level of inhibition of expression of the target gene. Forexample, if one nucleotide mismatch between the complementary nucleotidesequence of the complementary RNA strand and the correspondingnucleotide sequence of the target mutant gene provides optimalinhibition of expression (e.g., 99%), while two and three nucleotidemismatches provide medium and low levels of inhibition of expression(e.g., 60% and 20%), respectively, and four nucleotide mismatchesproduces no detectable inhibition of expression (0%), then a dsRNAcomprising a three-nucleotide mismatch would be chosen. In the otherhand, for example, if one nucleotide mismatch between the complementaryRNA strand and the target mutant gene provides optimal inhibition ofexpression (e.g., 95%), while two and three nucleotide mismatchesprovide low (e.g., 30%) or no detectable inhibition of expression (0%),respectively, then a dsRNA comprising a two-nucleotide mismatch would bechosen. Such levels provide some inhibition of expression of the targetgene, and hence some therapeutic value, but with minimal risk ofcross-reactivity with the normal cellular genes, and thus minimal sideeffects. The use of dsRNA having minimal risk of cross-reactivity,albeit sub-optimal efficacy, is particularly advantageous in situationswhere the side effects are unacceptable or severe.

The present inventors have also discovered that one can improve theefficiency of inhibition by modifying the degree of complementaritybetween the two RNA strands of the dsRNA. Such a finding is surprisingand unexpected, since reducing the level of complementarity between thehybridizing strands typically results in a decrease in stability, thusreducing the effectiveness of inhibition. One of skill in the art mayreadily determine the degree of mismatching that may be toleratedbetween any given RNA strands based upon the melting temperature and,therefore, the thermal stability of the resulting duplex. Determinationof the appropriate number and location of mismatches for a particulardsRNA can also be made using conventional cell-based assays or on thebasis of in vivo testing using an appropriate animal model, as describedelsewhere herein.

In another embodiment, at least one end of the dsRNA has asingle-stranded nucleotide overhang of between one and four, preferablyone or two nucleotides. dsRNAs having at least one nucleotide overhanghave unexpectedly superior inhibitory properties than their blunt-endedcounterparts. Moreover, the present inventors have discovered that thepresence of only one nucleotide overhang strengthens the interferenceactivity of the dsRNA, without affecting its overall stability. dsRNAhaving only one overhang has proven particularly stable and effective invivo, as well as in a variety of cells, cell culture mediums, blood, andserum. Preferably, the single-stranded overhang is located at the3′-terminal end of the complementary (antisense) RNA strand or,alternatively, at the 3′-terminal end of the second (sense) strand. ThedsRNA may also have a blunt end, preferably located at the 5′-end of thecomplementary (antisense) strand. Such dsRNAs have improved stabilityand inhibitory activity, thus allowing administration at low dosages,i.e., less than 5 mg/kg body weight of the recipient per day. The dsRNAmay have a blunt end, preferably located at the 5′-end of thecomplementary (antisense) strand. Preferably, the complementary strandof the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end isblunt. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate. In a particularlypreferred embodiment, the complementary (antisense) strand is 23nucleotides in length; the second (sense) strand is 21 nucleotides inlength; the 3′-end of the complementary (antisense) RNA strand has atwo-nucleotide overhang; and the end of the dsRNA at the 5′-terminal endof the complementary (antisense) strand is smooth.

In an optional embodiment, the dsRNA is chemically modified for improvedstability, i.e., enhanced resistance to degradation and/or stranddissociation. In this embodiment, the integrity of the duplex structureis strengthened by at least one, and preferably two, chemical linkages.Chemical linking may be achieved by any of a variety of well-knowntechniques, for example by introducing covalent, ionic or hydrogenbonds; hydrophobic interactions, van der Waals or stacking interactions;by means of metal-ion coordination, or through use of purine analogues.In one embodiment, the linker is a hexa-ethylene glycol linker. In thiscase, the dsRNAs are produced by solid phase synthesis and thehexa-ethylene glycol linker is incorporated according to standardmethods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996)35:14665-14670). In a preferred embodiment, the 5′-end of thecomplementary (antisense) RNA strand and the 3′-end of the second(sense) RNA strand are chemically linked via a hexa-ethylene glycollinker.

III. PHARMACEUTICAL COMPOSITIONS COMPRISING dsRNA

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a dsRNA, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising the dsRNA is useful for treating adisease or disorder associated with the expression a target mutant gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit expression of the targetmutant gene. The present inventors have found that, because of theirefficiency, compositions comprising the dsRNA of the invention can beadministered at surprisingly low dosages. A maximum dosage of 5 mg dsRNAper kilogram body weight per day is sufficient to inhibit or suppressexpression of the target gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, preferablyin the range of 0.1 to 200 micrograms per kilogram body weight per day,more preferably in the range of 0.1 to 100 micrograms per kilogram bodyweight per day, even more preferably in the range of 1.0 to 50micrograms per kilogram body weight per day, and most preferably in therange of 1.0 to 25 micrograms per kilogram body weight per day. Thepharmaceutical composition may be administered once daily, or the dsRNAmay be administered as two, three, four, five, six or more sub-doses atappropriate intervals throughout the day. In that case, the dsRNAcontained in each sub-dose must be correspondingly smaller in order toachieve the total daily dosage. The dosage unit can also be compoundedfor delivery over several days, e.g., using a conventional sustainedrelease formulation which provides sustained release of the dsRNA over aseveral day period. Sustained release formulations are well known in theart. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse models areavailable for hematopoietic malignancies such as leukemias, lymphomasand acute myelogenous leukemia. The MMHCC (Mouse models of Human CancerConsortium) web page (emice.nci.nih.gov), sponsored by the NationalCancer Institute, provides disease-site-specific compendium of knowncancer models, and has links to the searchable Cancer Models Database(cancermodels.nci.nih.gov), as well as the NCI-MMHCC mouse repository.Examples of the genetic tools that are currently available for themodeling of leukemia and lymphomas in mice, and which are useful inpracticing the present invention, are described in the followingreferences: Maru, Y., Int. J. Hematol. (2001) 73:308-322; Pandolfi, P.P., Oncogene (2001) 20:5726-5735; Pollock, J. L., et al., Curr. Opin.Hematol. (2001) 8:206-211; Rego, E. M., et al., Semin. in Hemat. (2001)38:4-70; Shannon, K. M., et al. (2001) Modeling myeloid leukemia tumorssuppressor gene inactivation in the mouse, Semin. Cancer Biol. 11,191-200; Van Etten, R. A., (2001) Curr. Opin. Hematol. 8, 224-230; Wong,S., et al. (2001) Oncogene 20, 5644-5659; Phillips J A., Cancer Res.(2000) 52(2):437-43; Harris, A. W., et al, J. Exp. Med. (1988)167(2):353-71; Zeng X X et al., Blood. (1988) 92(10):3529-36; Eriksson,B., et al., Exp. Hematol. (1999) 27(4):682-8; and Kovalchuk, A., et al.,J. Exp. Med. (2000) 192(8): 1183-90. Mouse repositories can also befound at: The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of dsRNA, as well as for determining a therapeuticallyeffective dose.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredients mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavoring agents, coloring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while corn starch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of dsRNA inthe cells that express the target gene. Such substances include, forexample, micellar structures, such as liposomes or capsids, as describedbelow. Surprisingly, the present inventors have discovered thatcompositions containing only naked dsRNA and a physiologicallyacceptable solvent are taken up by cells, where the dsRNA effectivelyinhibits expression of the target gene. Although microinjection,lipofection, viruses, viroids, capsids, capsoids, or other auxiliaryagents are required to introduce dsRNA into cell cultures, surprisinglythese methods and agents are not necessary for uptake of dsRNA in vivo.Aqueous suspensions according to the invention may include suspendingagents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the dsRNA against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

In one embodiment, the encapsulated formulation comprises a viral coatprotein. In this embodiment, the dsRNA may be bound to, associated with,or enclosed by at least one viral coat protein. The viral coat proteinmay be derived from or associated with a virus, such as a polyoma virus,or it may be partially or entirely artificial. For example, the coatprotein may be a Virus Protein 1 and/or Virus Protein 2 of the polyomavirus, or a derivative thereof.

The present inventors have discovered that there is a direct correlationbetween the number of complementary nucleotides within the dsRNA (i.e.,the number of base pairs between the two complementary RNA strands) andthe molecule's inhibitory activity. Reducing the complementarity betweenthe RNA strands of the dsRNA also generally reduces the intracellular orin vivo stability of the dsRNA. Thus, by modifying the number of basepairs within the dsRNA, one can typically adjust the efficiency ofinhibition of expression of the target gene.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs useful according to the invention can beadministered in combination with other known agents effective intreatment of diseases. In any event, the administering physician canadjust the amount and timing of dsRNA administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

For oral administration, the dsRNAs useful in the invention willgenerally be provided in the form of tablets or capsules, as a powder orgranules, or as an aqueous solution or suspension.

IV. METHODS FOR TREATING DISEASES CAUSED BY EXPRESSION OF A TARGET GENE

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of a target mutant gene. In this embodiment, the dsRNAcan act as novel therapeutic agents for controlling one or more ofcellular proliferative and/or differentiative disorders. The methodcomprises administering a pharmaceutical composition of the invention tothe patient (e.g., human), such that expression of the target mutantgene is silenced. Because of their high specificity, the dsRNAs of thepresent invention specifically target mRNAs of mutant genes of diseasedcells and tissues, as described elsewhere herein.

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the dsRNA can be brought intocontact with the cells or tissue exhibiting the disease. For example,dsRNA substantially identical to all or part of a mutated geneassociated with cancer, or one expressed at high levels in tumor cells,e.g. aurora kinase, may be brought into contact with or introduced intoa cancerous cell or tumor gene.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin. Asused herein, the terms “cancer,” “hyperproliferative,” and “neoplastic”refer to cells having the capacity for autonomous growth, i.e., anabnormal state of condition characterized by rapidly proliferating cellgrowth. These terms are meant to include all types of cancerous growthsor oncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. Proliferative disorders also include hematopoieticneoplastic disorders, including diseases involvinghyperplastic/neoplatic cells of hematopoietic origin, e.g., arising frommyeloid, lymphoid or erythroid lineages, or precursor cells thereof.

Mutations in cellular genes that directly or indirectly control cellgrowth and differentiation are considered to be the main cause ofcancer. There are approximately thirty families of genes, calledoncogenes, which are implicated in human tumor formation. Members of onesuch family, the RAS gene family, are carried in a broad range ofeukaryotes and are frequently found to be mutated in human tumors.Humans carry three functional RAS oncogenes, H-RAS, K-RAS, and N-RAS,coding for 21 kDa proteins 188-189 amino acids long (see, e.g., Lowy &Willumsen, Annu. Rev. Biochem. (1993) 2:851-891, 1993). RAS, H-RAS, andN-RAS have been detected in more human tumor types and at higherfrequencies than any other oncogenes (Bishop, Cell (1991) 64:235-248).In their normal state, proteins produced by the RAS genes are thought tobe involved in normal cell growth and maturation. Mutation of the RASgene, causing an amino acid alteration at one of three criticalpositions in the protein product, results in conversion to a form thatis implicated in tumor formation. Over 90% of pancreaticadenocarcinomas, about 50% of prostate cancers, about 50% of adenomasand adenocarcinomas of the colon, about 50% of adenocarcinomas of thelung, about 50% of carcinomas of the thyroid, about 25% of melanomas,and a large fraction of malignancies of the blood, such as acute myeloidleukemia and myelodysplastic syndrome, have been found to containactivated RAS oncogenes.

Mammalian genes frequently acquire transformation-inducing properties bysingle point mutations within their coding sequences. Mutations innaturally occurring RAS oncogenes have been localized to codons 12, 13,and 61 (Gibbs, et al., Proc. Natl. Acad. Sci. USA (1988) 85:5026-5030).These mutant forms remain in the active GTP form much longer than thewild-type, and presumably, the continual transmission of a signal by themutant forms is responsible for their oncogenic properties. Reducing thelevel of K-RAS gene expression might inhibit proliferation or reversetransformation in malignant cells transformed by mutated K-RAS (Georges,et al., Cancer Res. 53:1743-1746, 1993; Mukhopadhyay et al., Cancer Res.(1991) 51:1744-1748; Kashani-Sabet, et al., Cancer Res. (1994)54:900-902; and Aoki et al., Cancer Res. (1995) 55:3810-3815).

In addition to the foregoing oncogenes, the methods and compositions ofthe invention can be applied to other disease-related target geneshaving a point mutation. Gene mutations have been reported in more than1000 different human genes. Data on these mutations and their associatedphenotypes have been collated and are available online through two majordatabases: Online Mendelian Inheritance in Man in Baltimore and theHuman Gene Mutation Database in Cardiff. For example, there is a highfrequency of CG to TG or CA mutations in the human genome due todeamination of 5′ methyl-cytosine. Short deletions or insertions of lessthan 20 nucleotides are also very common mutations in humans. See, e.g.,Antonarakis, S. E., Eur. Pediatr. (2000)159(3):5173-8.

Sachidanandam et al. describes a map of human genome sequence variationcontaining 1.42 million single nucleotide polymorphisms, which is usefulfor identifying biomedically important genes for diagnosis and therapy(Sachidanandam, R., et al., Nature (2001) 409(6822):821-2 and Nature(2001) 409(6822):822-3). The map integrates all publicly available SNPswith described genes and other genomic features. An estimated 60,000SNPs fall within exon (coding and untranslated regions), and 85% ofexons are within 5 kb of the nearest SNP. Clifford et al. providesexpression-based genetic/physical maps of single-nucleotidepolymorphisms identified by the cancer genome anatomy project (Clifford,R., et al., Genome Res (2000) 10(8):1259-65). In addition to SNP maps,Sachidanandam et al. provide maps containing SNPs in genes expressed inbreast, colon, kidney, liver, lung, or prostate tissue. The integratedmaps, a SNP search engine, and a Java-based tool for viewing candidateSNPs in the context of EST assemblies can be accessed via the CGAP-GAIweb site http://cgap.nci.nih.gov/GAI/).

The Human Gene Mutation Database (HGMD) represents a comprehensive corecollection of data on published germline mutations in nuclear genesunderlying human inherited disease. The data is publicly available athttp://uwcm.ac.uk/uwcm/mg/hgrnd0.html. Integration with phenotypic,structural and mapping information are available through links betweenHGMD and both the Genome Database (GDB) and Online Mendelian Inheritancein Man (OMIM), Baltimore, USA. Hypertext links have also beenestablished to Medline abstracts through Entrez, and to a collection of458 reference cDNA sequences also used for data checking See, e.g.,Krawczak, M, and D. N. Cooper, Genet. (1997) 13(3):121-2; and Cooper, D.N., et al., Nucleic Acids Res (1998) 26(1):285-7. Keio Mutation Database(KMDB) is also a database of mutations in human disease-causing genes.These KMDBs are accessible through http://mutview.dmb.med.keio.ac.jpwith advanced internet browsers.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Inpreferred embodiments, the pharmaceutical compositions are administeredby intravenous or intraparenteral infusion or injection.

V. METHODS FOR INHIBITING EXPRESSION OF A MUTANT GENE

In yet another aspect, the invention relates to a method for inhibitingthe expression of a mutant gene in an organism. The method comprisesadministering a composition of the invention to the organism such thatexpression of the mutant gene is silenced as compared to thecorresponding wild-type gene. The organism may be an animal or a plant.Because of their high specificity, the dsRNAs of the present inventionspecifically target RNAs (primary or processed) of target mutant genes,and at surprisingly low dosages. Compositions and methods for inhibitingthe expression of a target gene using dsRNAs can be performed asdescribed elsewhere herein.

In one embodiment, the invention comprises administering a compositioncomprising a dsRNA, wherein the dsRNA comprises a nucleotide sequencewhich is substantially complementary to an RNA transcript of the targetmutant gene and partially complementary to the corresponding wild-typegene. When the organism to be treated is a mammal, such as a human, thecomposition may be administered by any means known in the art including,but not limited to oral or parenteral routes, including intravenous,intramuscular, intraperitoneal, subcutaneous, transdermal, airway(aerosol), rectal, vaginal and topical (including buccal and sublingual)administration. In preferred embodiments, the compositions areadministered by intravenous or intraparenteral infusion or injection.

The methods for inhibition the expression of a target gene can beapplied to any mutant gene one wishes to silence, thereby selectivelyinhibiting its expression. Examples of human genes which can be targetedfor silencing include, without limitation, an oncogene; cytokinin gene;idiotype protein gene (Id protein gene); prion gene; gene that expressesmolecules that induce angiogenesis, adhesion molecules, and cell surfacereceptors; genes of proteins that are involved in metastasizing and/orinvasive processes; genes of proteases as well as of molecules thatregulate apoptosis and the cell cycle; and genes that express the EGFreceptor; the multi-drug resistance 1 gene (MDR1 gene).

The methods for inhibition the expression of a target mutant gene canalso be applied to any plant gene one wishes to silence, therebyspecifically inhibiting its expression.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

EXAMPLES Example 1 Effect of Complementarity on Inhibition of Expressionof Mutant Target Gene

In order to make a reporter system, a 26-nucleotide-long sequence of acDNA sequence that serves as a target gene for the 3′-untranslatedregion of a corresponding HCV-RNA was fused with the open reading frameof the luciferase gene from the pGL3 expression vector. The pGL3expression vector came from Promega Co., and is registered under GeneAccession No. U47296 with the National Center for BiotechnologyInformation (NCBI), National Library of Medicine, Building 38A,Bethesda, Md. 20894. Nucleotides 280 to 1932 were used as the luciferasegene. The 26-nucleotide-long sequence is one that is frequentlypreserved in many HCV genomes and their subtypes. The 26 nucleotides ofthe HCV genome that is registered with the NCBI under Gene Accession No.D89815 correspond to Nucleotides 9531 to 9556. They exhibit thefollowing sequence (mutant target gene):

(SEQ ID NO: 1) 5′-gtcacggct agctgtgaaa ggtccgt-3′.

The resulting fusion gene was cloned as a Bam Hi/Not I DNA fragment inthe eukaryote expression plasmid pcDNA 3.1 (+) by Invitrogen GmbH,Karlsruhe Technology Park, Emmy Noether Str. 10, 76131 Karlsruhe,Catalogue No. V790-20. The resulting plasmid was designated p8. ThepCMVβGal plasmid from Clontech, Gene Accession No. U13186, NCBI, whichencodes the enzyme β-galactosidase was used as a control fortransfection efficiency. The fusion gene plasmid, pCMVβGal, and thevarious dsRNAs were co-transfected into cells of the liver cell lineHuH-7 (JCRB0403, Japanese Collection of Research Bioresources Cell Bank,National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku,Tokyo 158, Japan). Inhibition of expression of the luciferase gene thatwas induced by the dsRNAs was determined in relation to the expressionof the β-galactosidase gene.

The dsRNAs have the following sequences (designated as SEQ ID NO:2 toSEQ ID NO:13 in the sequence protocol):

-   -   HCV1+2, whose 51 strand is completely complementary to the HCV        sequence in the fused HCV-luciferase gene:

S2:    5′-ACG GCU AGC UGU GAA AGG UCC GU-3′ (SEQ ID NO: 2) S1:3′-AG UGC CGA UCG ACA CUU UCC AGG-5′ (SEQ ID NO: 3)

-   -   HCV3+4, which is complementary neither to the HCV—nor to the        luciferase sequence in the fused HCV-luciferase gene, and serves        as the negative control:

S2:    5′-AGA CAG UCG ACU UCA GCC U GG-3′ (SEQ ID NO: 12) S1:3′-GG UCU GUC AGC UGA AGU CGG A-5′ (SEQ ID NO: 13)

-   -   HCV5+6, whose S1 strand is complementary to the HCV sequence in        the fused HCV-luciferase gene, except for the nucleotides that        are in bold:

S2:    5′-ACG GCU AGC UGU GAA UGG UCC GU-3′ (SEQ ID NO: 6) S1:3′-AG UGC CGA UCG ACA CUU ACC AGG-5′ (SEQ ID NO: 7)

-   -   HCV7+8, whose S1 strand is complementary to the HCV sequence in        the fused HCV-luciferase gene, except for the two nucleotides        that are in bold:

S2:    5′-ACG GCA AGC UGU GAA UGG UCC GU-3′ (SEQ ID NO: 8) S1:3′-AG UGC CGU UCG ACA CUU ACC AGG-5′ (SEQ ID NO: 9)

-   -   Luc1+2, whose S1 strand is completely complementary to a        luciferase sequence in the fused HCV-luciferase gene, and which        serves as the positive control:

S2:    5′-CGU UAU UUA UCG GAG UUG CAG UU-3′ (SEQ ID NO: 10) S1:3′-GC GCA AUA AAU AGC CUC AAC GUC-5′ (SEQ ID NO: 11)

-   -   K3s+K3 as, which is complementary neither to the HCV—nor to the        luciferase sequence in the fused HCV-luciferase gene, and which        serves as the negative control:

S2:   5′-G AUG AGG AUC GUU UCG CAU GA-3′ (SEQ ID NO: 4) S 1:3′-UCC UAC UCC UAG CAA AGC GUA-5′ (SEQ ID NO: 5)

-   -   HCV5+2, whose S1 strand is completely complementary to the HCV        sequence, and whose S2 strand is complementary to the HCV        sequence in the fused HCV-luciferase gene, except for the        nucleotides that are in bold:

S2:    5′-ACG GCU AGC UGU GAA UGG UCC GU-3′ (SEQ ID NO: 6) S1:3′-AG UGC CGA UCG ACA CUU UCC AGG-5′ (SEQ ID NO: 3)

-   -   HCV1+6, whose S2 strand is completely complementary to the HCV        sequence, and whose S1 strand is complementary to the HCV        sequence in the fused HCV-luciferase gene, except for the        nucleotides that are in bold:

S2:    5′-ACG GCU AGC UGU GAA AGG UCC GU-3′ (SEQ ID NO: 2) S1:3′-AG UGC CGA UCG ACA CUU ACC AGG-5′ (SEQ ID NO: 7)

HuH-7 cells were cultured in DMEM with 10% FCS. In preparation fortransfection, 2×10⁴ cells were seeded per well of a 96-well cell cultureplate. The cells were transfected 24 hours after seeding with 110 μltransfection medium per well total volume. Each transfection wasrepeated three times. 3 μg of the PCMVβGal plasmid and 1 μg of the p8plasmid were added to the transfection mixture. The transfection mediumcontained 0.25 μg of the plasmid mixture per well, and 200, 100, 50, 25,12.5, or 0 nmol/l of the aforementioned dsRNAs. “Gene Porter 2” fromPEQLAB Biotechnology GmbH, Carl Thiersch Str. 2b, 91052 Erlangen,Catalogue No. 13-T202007 was used for the transfection in accordancewith manufacturer's instructions. Transfected cells were incubated at37° C. and 5% CO₂. One day after transfection, 35 μl of fresh medium wasadded per well, and the cells were incubated for another 24 hours. Theeffect of the dsRNAs was determined by quantifying expressedβ-galactosidase with the “Galacto-Star” from Tropix Corp., 47 WigginsAvenue, Bedford, Mass. 01730, Catalogue No. BM100S, and the effect onthe expressed luciferase was determined by chemoluminescence reactionwith “Luciferase” from Tropix, Catalogue No. BC100L. The cell lysate wasmade in accordance with manufacturer's instructions, and 2 μl of it wasused per analysis to test for β-galactosidase and 5 μl was used peranalysis to test for luciferase. Chemoluminescence measurements weredetermined using a Sirius Luminometer (Berthold Detection Systems GmbH,Bleichstr. 56-68, 75173 Pforzheim, Germany). The relative activity ofluciferase as a measurement of expression was determined by dividing theluciferase value by the β-galactosidase value. An average was thencalculated for the three assays. The average for cells transfectedwithout dsRNA was arbitrarily defined as 1.0. The other averages wereexpressed as a ratio with that value, and these were depictedgraphically in FIGS. 1 and 2.

Luc1+2 (positive control) led to the most marked inhibition ofluciferase activity (FIGS. 1 and 2). In the presence of HCV1+2, whichwas completely complementary to the target sequence for the reporterplasmid, a clear reduction in luciferase activity was also discernible(FIGS. 1 and 2). Luciferase activity increased with decreasingconcentrations of HCV1+2. The HCV5+6, which is not complementary to thetarget sequence by one nucleotide, is approximately as effective ininhibiting luciferase as HCV1+2, particularly at low concentrations(FIG. 1).

HCV7+8 inhibits expression of luciferase both at high and at lowconcentrations only to the same degree as the negative controls HCV3+4and K3S+K3AS (FIGS. 1 and 2). The scant inhibition of luciferaseactivity is to be seen as a nonspecific effect. As far as thespecificity of this dsRNA is concerned, dsRNA need be eithercomplementary to the target gene, or off by only one nucleotide in orderto inhibit expression of the specific target gene as compared toexpression of the original gene.

In HCV5+2, one nucleotide in the S2 sense strand is not complementary tothe S1 antisense strand, although the S1 antisense strand is completelycomplementary to the target gene. This dsRNA is as effective as LUC1+2and HCV1+2 (FIGS. 1 and 2). In HCV6+1, one nucleotide in the S1antisense strand is not complementary to the S2 sense strand, while theS1 antisense strand is not complementary to the target gene by onenucleotide. HCV6+1 inhibits expression less effectively than HCV5+6, butmore effectively and HCV7+8 (FIG. 2). In other words, specificity andeffectiveness of the expression-inhibiting action of dsRNA depends moreon the sequence of the S1 antisense strand than on that of the S2 sensestrand.

HCV3+4 (FIG. 1) and K3S+K3AS (FIG. 2), which serve as the negativecontrols, led to no and little inhibition of luciferase activity,respectively. The minor inhibition is nonspecific, as it is notdependent on the dsRNA concentrations used. The data show that at leasttwo nucleotides in the antisense strand of a dsRNA that are notcomplementary to an original gene are necessary to prevent inhibition ofexpression of the original gene. Furthermore, the data show that it ispossible to modify the effectiveness of inhibition of the expression ofthe dsRNA by lessening the extent of complementarity of the singlestrands that form the dsRNA.

Example 2 Treatment of a Colon Cancer Patient with K-Ras siRNA

In this Example, a K-Ras double stranded siRNA directed against K-Rastranscripts with a mutation at codon 12, is injected into colon cancerpatients and shown to specifically inhibit K-Ras gene expression.

SiRNA Synthesis

siRNAs (K-Ras) directed against mutant K-Ras mRNA are chemicallysynthesized with or without a hexaethylene glycol linker.

Synthesis and Preparation of dsRNAs

Oligoribonucleotides are synthesized with an RNA synthesizer (Expedite8909, Applied Biosystems, Weiterstadt, Germany) and purified by HighPressure Liquid Chromatography (HPLC) using NucleoPac PA-100 columns,9×250 mm (Dionex Corp.; low salt buffer: 20 mM Tris, 10 mM NaClO₄, pH6.8, 10% acetonitrile; the high-salt buffer was: 20 mM Tris, 400 mMNaClO4, pH 6.8, 10% acetonitrile. flow rate: 3 ml/min). Formation ofdouble stranded siRNAs is then achieved by heating a stoichiometricmixture of the individual complementary strands (10 M) in 10 mM sodiumphosphate buffer, pH 6.8, 100 mM NaCl, to 80-90° C., with subsequentslow cooling to room temperature over 6 hours,

In addition, dsRNA molecules with linkers may be produced by solid phasesynthesis and addition of hexaethylene glycol as a non-nucleotide linker(D. Jeremy Williams, Kathleen B. Hall, Biochemistry, 1996, 35,14665-14670). A Hexaethylene glycol linker phosphoramidite (ChruachemLtd, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, G20OUA, Scotland, UK) is coupled to the support bound oligoribonucleotideemploying the same synthetic cycle as for standard nucleosidephosphoramidites (Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg,Germany) but with prolonged coupling times. Incorporation of linkerphosphoramidite is comparable to the incorporation of nucleosidephosphoramidites.

siRNA Administration and Dosage

The present example provides for pharmaceutical compositions for thetreatment of human colon cancer patients comprising a therapeuticallyeffective amount of a K-Ras siRNAs, in combination with apharmaceutically acceptable carrier or excipient. Examples of suitablecarriers are found in standard pharmaceutical texts, e.g. “Remington'sPharmaceutical Sciences”, 16th edition, Mack Publishing Company, Easton,Pa., 1980.

The dosage of the siRNAs will vary depending on the form ofadministration. In the case of an intravenous injection, thetherapeutically effective dose of siRNA per injection is in a dosagerange of approximately 1-500 microgram/kg body weight per day,preferably at a dose of 100 micrograms/kg/body weight per day. Inaddition to the active ingredient, the compositions usually also containsuitable buffers, for example phosphate buffer, to maintain anappropriate pH and sodium chloride, glucose or mannitol to make thesolution isotonic. The administering physician will determine the dailydosage which will be most suitable for an individual and will vary withthe age, gender, weight and response of the particular individual, aswell as the severity of the patient's symptoms. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention. The siRNAs of the present inventionmay be administered alone or with additional siRNA species directed toK-Ras mutant transcripts having other activating point mutations or incombination with other pharmaceuticals.

Efficacy of K-Ras siRNA Treatment

Efficacy of the siRNA treatment is determined at defined intervals afterthe initiation of treatment using PCR on total RNA extracted from tumorbiopsies. Cytoplasmic RNA from the tumor biopsy, taken prior to andduring treatment, is purified with the help of the RNeasy Kit (Qiagen,Hilden) and mutant K-Ras mRNA levels are quantitated by RT-PCR (see WO96/15262 for description of PCR protocol for the detection of mutantK-Ras gene expression).

1. A double-stranded ribonucleic acid (dsRNA) for selective inhibitionof expression of a mutant target gene, the dsRNA comprising: a firststrand that has a complementary region that is complementary to at leasta portion of an RNA transcript of at least part of the mutant targetgene; and a second strand of the dsRNA, wherein the first and secondstrands are complementary or substantially complementary to each other,wherein the first and second strands of the dsRNA are 19 to less than 30nucleotides in length, wherein the complementary region of the firststrand of the dsRNA contains a mismatch relative to the mutant targetgene and at least one more mismatch relative to a wild type form of themutant target gene, and wherein the dsRNA selectively inhibitsexpression of the mutant target gene in a mammalian cell relative to thewild-type form of the mutant target gene.
 2. The dsRNA of claim 1,wherein the mismatch relative to the mutant target gene is at least 2nucleotides from an end of the first strand.
 3. The dsRNA of claim 1,wherein the mutant target gene comprises a point mutation that is absentfrom the wild-type form of the target gene.
 4. The dsRNA of claim 1,wherein the first strand comprises a nucleotide mismatch with the secondstrand.
 5. The dsRNA of claim 1, wherein the first and second strandsare 23 nucleotides in length.
 6. The dsRNA of claim 1, wherein the firstand second strands are 21 nucleotides in length.
 7. The dsRNA of claim1, wherein the first and second strands of the dsRNA are 19 to 24nucleotides in length.
 8. The dsRNA of claim 1, wherein the mutanttarget gene is an oncogene.
 9. The dsRNA of claim 1, wherein selectivelyinhibiting expression further comprises the dsRNA having a substantiallygreater inhibitory effect on expression of the mutant target gene in thecell than on the wild-type form of the mutant target gene.
 10. The dsRNAof claim 1, wherein the second strand of the dsRNA is from 1 to 4nucleotides longer than the first strand such that the dsRNA has a 1 to4 nucleotide single stranded region at either the 5′ or 3′ end of thesecond strand.
 11. The dsRNA of claim 1, wherein the second strand ofthe dsRNA is from 1 to 4 nucleotides longer than the first strand suchthat the dsRNA has a 1 to 4 nucleotide single stranded region at the 3′end of the second strand.
 12. The dsRNA of claim 1, wherein the secondstrand of the dsRNA is from 1 to 2 nucleotides longer than the firststrand such that the dsRNA has a 1 to 2 nucleotide single strandedregion at either the 5′ or 3′ end of the second strand.
 13. The dsRNA ofclaim 1, wherein the second strand of the dsRNA is from 1 to 2nucleotides longer than the first strand such that the dsRNA has a 1 to2 nucleotide single stranded region at the 3′ end of the second strand.14. A pharmaceutical composition for selectively inhibiting theexpression of a mutant target gene in a mammal, the compositioncomprising: a dsRNA and pharmaceutically acceptable carrier, wherein thedsRNA comprises: a first strand that has a complementary region that iscomplementary to at least a portion of an RNA transcript of at leastpart of the mutant target gene; and a second strand of the dsRNA,wherein the first and second strands are complementary or substantiallycomplementary to each other, wherein the first and second strands of thedsRNA are 19 to less than 30 nucleotides in length, wherein thecomplementary region of the first strand of the dsRNA contains amismatch relative to the mutant target gene and at least one moremismatch relative to a wild type form of the mutant target gene, andwherein the dsRNA selectively inhibits expression of the mutant targetgene in a mammalian cell relative to the wild-type form of the mutanttarget gene.
 15. The pharmaceutical composition of claim 14, wherein themismatch relative to the mutant target gene is at least 2 nucleotidesfrom an end of the first strand.
 16. The pharmaceutical composition ofclaim 14, wherein the mutant target gene comprises a point mutation thatis absent from the wild-type form of the target gene.
 17. Thepharmaceutical composition of claim 14, wherein the first strandcomprises a nucleotide mismatch with the second strand.
 18. Thepharmaceutical composition of claim 14, wherein the first and secondstrands are 23 nucleotides in length.
 19. The pharmaceutical compositionof claim 14, wherein the first and second strands of the dsRNA are 19 to24 nucleotides in length.
 20. The pharmaceutical composition of claim14, wherein the mutant target gene is an oncogene.
 21. Thepharmaceutical composition of claim 14, wherein selectively inhibitingexpression further comprises the dsRNA having a substantially greaterinhibitory effect on expression of the mutant target gene in the cellthan on the wild-type form of the mutant target gene.
 22. Thepharmaceutical composition of claim 14, wherein the second strand of thedsRNA is from 1 to 2 nucleotides longer than the first strand such thatthe dsRNA has a 1 to 2 nucleotide single stranded region at either the5′ or 3′ end of the second strand.
 23. The pharmaceutical composition ofclaim 14, wherein the second strand of the dsRNA is from 1 to 2nucleotides longer than the first strand such that the dsRNA has a 1 to2 nucleotide single stranded region at the 3′ end of the second strand.24. The pharmaceutical composition of claim 14, wherein thecomplementary RNA strand is complementary to an RNA transcript of thetarget mutant gene.
 25. The pharmaceutical composition of claim 14,wherein the dosage unit of dsRNA is in a range of 0.01 to 5.0milligrams, 0.01 to 2.5 milligrams, 0.1 to 200 micrograms, 0.1 to 100micrograms, 1.0 to 50 micrograms, or 1.0 to 25 micrograms per kilogrambody weight of the mammal.
 26. The pharmaceutical composition of claim14, wherein the dosage unit of dsRNA is in a range of 1.0 to 25micrograms per kilogram body weight of the mammal.
 27. Thepharmaceutical composition of claim 14, wherein the mammal is a human.28. The pharmaceutical composition of claim 14, wherein thepharmaceutically acceptable carrier is an aqueous solution.
 29. Thepharmaceutical composition of claim 28, wherein the aqueous solution isphosphate buffered saline.
 30. The pharmaceutical composition of claim14, wherein the pharmaceutically acceptable carrier comprises a micellarstructure selected from the group consisting of: a liposome, capsid,capsoid, polymeric nanocapsule, and polymeric microcapsule.
 31. Thepharmaceutical composition of claim 30, wherein the polymericnanocapsule and polymeric microcapsule comprise polybutylcyanoacrylate.32. The pharmaceutical composition of claim 14, which is formulated tobe administered by inhalation, infusion, injection, or orally.
 33. Thepharmaceutical composition of claim 14, which is formulated to beadministered by intravenous or intraperitoneal injection.